Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA.
Nucleic Acids Res. 2020 Jun 4;48(10):5788-5798. doi: 10.1093/nar/gkaa329.
The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.
CRISPR-Cas12a 是一种具有 RNase 和 DNase 活性的 II 类、V 型簇状规则间隔短回文重复（CRISPR）系统。与 CRISPR-Cas9 系统相比，它识别富含 T 的 PAM 序列，具有多重基因组编辑的优势。在这里，在裂殖酵母 Schizosaccharomyces pombe 中，我们成功地实现了 CRISPR-Cas12a 系统用于多功能基因组编辑和操作。除了 rrk1 启动子外，我们还使用了来自内源性编码基因的新 pol II 启动子来表达 Cas12a 的 crRNA，从而获得了更高的编辑效率。这种新设计扩展了启动子选择，可潜在应用于裂殖酵母和其他生物。此外，我们使用强组成型 pol II 启动子表达了 gRNA 阵列。该阵列转录本被 Cas12a 自身加工，释放出多个成熟的 crRNA。使用该构建体，可通过单个酵母转化实现多达三个基因座的多重基因组编辑。我们还构建了一种 CRISPR 干扰系统，使用无 DNase 活性的 Cas12a 显著抑制内源性基因表达。我们的研究为裂殖酵母中高效快速的基因组基因编辑和调控提供了第一个 CRISPR-Cas12a 工具包。
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